Efficient construction of long DNA duplexes with internal non-Watson–Crick motifs and modifications

AUTOR(ES)

Zheng, Xiaofeng

FONTE

Oxford University Press

RESUMO

We have developed a semi-synthetic approach for preparing long stretches of DNA (>100 bp) containing internal chemical modifications and/or non-Watson–Crick structural motifs which relies on splint-free, cell-free DNA ligations and recycling of side-products by non-PCR thermal cycling. A double-stranded DNA PCR fragment containing a polylinker in its middle is digested with two restriction enzymes and a small insert (∼20 bp) containing the modification or non-Watson–Crick motif of interest is introduced into the middle. Incorrect products are recycled to starting materials by digestion with appropriate restriction enzymes, while the correct product is resistant to digestion since it does not contain these restriction sites. This semi-synthetic approach offers several advantages over DNA splint-mediated ligations, including fewer steps, substantially higher yields (∼60% overall yield) and ease of use. This method has numerous potential applications, including the introduction of modifications such as fluorophores and cross-linking agents into DNA, controlling the shape of DNA on a large scale and the study of non-sequence-specific nucleic acid–protein interactions.

Documentos Relacionados

• Characterization of imperfect DNA duplexes containing unpaired bases and non-Watson-Crick base pairs.
• A curved RNA helix incorporating an internal loop with G·A and A·A non-Watson–Crick base pairing
• SURVEY AND SUMMARY: The non-Watson–Crick base pairs and their associated isostericity matrices
• Protonation of non-Watson–Crick base pairs and encapsidation of turnip yellow mosaic virus RNA
• Homologous recognition promoted by RecA protein via non-Watson-Crick bonds between identical DNA strands.